Characterization of North American Armillaria species: genetic relationships determined by ribosomal DNA sequences and AFLP markers

Phylogenetic and genetic relationships among 10 North American Armillaria species were analysed using sequence data from ribosomal DNA (rDNA), including intergenic spacer (IGS‐1), internal transcribed spacers with associated 5.8S (ITS + 5.8S), and nuclear large subunit rDNA (nLSU), and amplified fragment length polymorphism (AFLP) markers. Based on rDNA sequence data, the nLSU region is less variable among Armillaria species than the ITS + 5.8S and IGS‐1 regions (nLSU < ITS + 5.8S < IGS‐1). Phylogenetic analyses of the rDNA sequences suggested Armillaria mellea, A. tabescens and A. nabsnona are well separated from the remaining Armillaria species (A. ostoyae, A. gemina, A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes). Several Armillaria species (A. calvescens, A. sinapina, A. gallica, NABS X and A. cepistipes) clustered together based on rDNA sequencing data. Based on the isolates used in this study, it appears that techniques based on IGS‐1, ITS + 5.8S, and/or D‐domain/3′ ends of nLSU are not reliable for distinguishing A. calvescens, A. sinapina, A. gallica and A. cepistipes. However, AFLP data provided delineation among these species, and AFLP analysis supported taxonomic classification established by conventional methods (morphology and interfertility tests). Our results indicate that AFLP genetic markers offer potential for distinguishing currently recognized North American Biological Species (NABS) of Armillaria in future biological, ecological and taxonomic studies.     

Identification of European Armillaria species by restriction-fragment-length polymorphisms of ribosomal DNA

Identification of European Armillaria species using ribosomal DNA (rDNA) restriction-fragment-length polymorphisms (RFLPs) was studied. A total of 44 Armillaria isolates representing five European biological species were examined. Whole-cell DNAs were digested with one or two (double digest) restriction endonucleases and probed with a cloned plasmid carrying one complete rDNA repeat copy of Saccharomyces carlsbergensis. Applying the restriction endonuclease Bgl II in combination with either Eco RI, Bam Hl or Hin dIII, it was possible to detect rDNA RFLPs allowing differentiation between all five European Armillaria species investigated in the study. The most conclusive results were obtained in the rDNA/Ava II RFLP pattern. All biological species showed unique Ava II banding patterns. Generally speaking, the interspecific similarities were around 42% and lower, indicating a distinct species separation. The analysis of rDNA RFLP patterns by a restriction-enzyme-rDNA-probe combination (for instance, Ava II or Bgl II/Hin dIII; probe pMY 60) is a practical means of identifying European Armillaria species for further taxonomic, phylogenetic and host-pathogen interaction studies.     

A rapid and holistic approach to screen susceptibility of Prunus species to Armillaria root rot

We describe a holistic in vitro technique for inoculating roots of Prunus species with Armillaria solidipes, which is faster and more successful than previous methods. This method allows associated active and passive host defences to be assessed. Sterile root segments of three Prunus spp. were placed next to or on top of 14‐day‐old cultures of A. solidipes. At 21 day, the success of fungal penetration and length of the fungal colonization, and host responses were evaluated. Also, an anti‐Armillaria and anti‐Cladosporium activity detection assays were conducted by utilizing the root periderm, the first tissue that needs to be penetrated by fungus. These methods revealed the variation in the relative tolerance of three Prunus spp. to A. solidipes. The overall success of fungal colonization in the wounded and intact root, host defence, and antifungal activity significantly differed among three Prunus spp. Results indicate that this in vitro method can be used as a preliminary step in screening tree species to Armillaria spp.     

Development and verification of a diagnostic assay based on EF‐1 α for the identification of Armillaria species in Northern Europe

The overall aim of this study was to develop a new, reliable and rapid diagnostic assay for differentiating six European Armillaria species based on variation in their elongation factor‐1 alpha (EF‐1 α) gene sequences and to verify a set of species‐specific primers on 61 Armillaria isolates from Europe. Partial sequences of the EF‐1 α gene obtained in Armillaria borealis, Armillaria cepistipes, Armillaria gallica, Armillaria mellea, Armillaria ostoyae and Armillaria tabescens revealed sufficient interspecific variation to distinguish among species using nested primers. These primers gave unambiguous bands when tested on representative isolates of five of these species. However, the EF‐1 α sequences of European A. borealis isolates clustered into two distinct clades, termed here AbX and AbY. Specific primers were subsequently designed and tested successfully on both AbX‐type and AbY‐type A. borealis isolates. The taxonomy of A. borealis needs to be elucidated to determine whether a new, as yet unnamed Armillaria taxon exists in Europe. Three A. borealis isolates were also found to have heterozygous sites in their EF‐1 α sequences, which suggests that the gene could exist in more than one copy or that these isolates contain hybrid sequences. A pyrosequencing method was also developed, targeting a small region of EF‐1 α intron 4, which was able to differentiate European Armillaria isolates to the species level and additionally could distinguish AbX‐type and AbY‐type A. borealis isolates.     

Distribution of Armillaria species in upland Ozark Mountain forests with respect to site,overstory species composition and oak decline

Armillaria root disease is a contributing factor to oak decline in the Ozark Mountains of central USA. We have identified Armillaria gallica, Armillaria mellea, and Armillaria tabescens in Quercus‐Carya‐Pinus forests of the region. Presence/absence patterns of each Armillaria species as well as all possible Armillaria species combinations were analysed by contingency tables and/or stepwise logistic multiple regressions with principal characteristics of the studied sites and forest stands, both quantitative and qualitative: geographic land‐type association, bedrock type, landform position, slope direction (aspect), soil type and soil surface stone cover, down woody debris, abundance and basal area of woody vegetation and decline mortality by species. Most decline mortality consisted of two red oak species (section Erythrobalanus, Quercus coccinea and Quercus velutina), which also were most sensitive to Armillaria infection. Site characteristics related to the distributions of Armillaria species and decline mortality were also related to the preponderance of Q. coccinea and Q. velutina, regional vegetation history (i.e. conversion of Pinus echinata stands to hardwoods), and the different strategies of territory acquisition and spread of the Armillaria species involved. The presence of A. gallica may reduce the activity of more virulent Armillaria species.     

Initial characterization of an unidentified Armillaria isolate from Serbia using LSU‐IGS1 and TEF‐1‐α genes

Armillaria species have a global distribution and play variable ecological roles, including causing root disease of diverse forest, ornamental and horticultural trees. Accurate identification of Armillaria species is critical to understand their distribution and ecological roles. This work focused on characterizing an unidentified Armillaria isolate from a Serbian forest using pairing, sequencing of the partial large subunit and intergenic spacer‐1 regions of rDNA (LSU‐IGS1) and the translation elongation factor‐1 alpha gene (tef‐1α) genes, and phylogenetic analyses. Despite previously obtained LSU‐IGS1 RFLP patterns that matched the newly described North American Armillaria altimontana, pairing tests and phylogenetic analyses of LSU‐IGS1 and tef‐1α sequences clearly demonstrate that the unidentified isolate is not A. altimontana. Based on LSU‐IGS1, Armillaria gallica isolates were polyphyletic, and the Serbian isolate clustered with a subset of European A. gallica isolates within a well‐supported clade (99%). Based on tef‐1α, the Serbian isolate appeared as a separate, well‐supported clade (97%) that was basal to other poorly resolved, polyphyletic clades containing European A. gallica isolates. It is speculated that the unidentified Armillaria isolate from Serbia could represent an evolutionary ancestral state because of its separate, basal position compared with other clades comprising polyphyletic European A. gallica isolates. Alternatively, this unidentified Serbian isolate could represent an unusual hybrid because of its high‐level sequence heterogeneity, represented by multiple two‐nucleotide codes, within tef‐1α. Further characterization is needed to confirm the taxonomic status and ecological/evolutionary significance of this unique, unknown Armillaria isolate from Serbia.     

Identification and distribution of Armillaria species associated with an oak decline event in the Arkansas Ozarks

Forests in the Ozark Mountains of northern Arkansas recently experienced a widespread oak decline event. Armillaria, a root rot fungus, has been associated with other oak decline events and may have been an important contributing factor to tree mortality in this event. Although Armillaria has been identified from the Ozark Mountains in Missouri, it has never been investigated in the Arkansas Ozarks. Molecular diagnostic techniques were used in this study to identify species of Armillaria present on roots removed from dead trees of two common oak species, northern red oak, Quercus rubra L., and white oak, Q. alba L., from three geographic areas and on three topographic positions – ridges, south‐ and west‐facing benches. Armillaria(A. mellea, A. gallica or A. tabescens) was identified from 31% of root samples taken from 102 trees in seven of nine sample plots. Armillaria mellea, occurred most often (20 samples, both oak species on seven plots) followed by A. gallica (10 samples, northern red oak only on four plots), and A. tabescens occurred twice (on northern red oak in a single plot). Thus, all three Armillaria species occurred on northern red oaks while A. mellea was the only species recovered from white oaks. Results varied by topographic position with samples from tree roots on ridges having the fewest positive identifications, one of 29. West‐facing benches had the highest positive samples with 20 of 41 testing positive and trees on south‐facing benches were intermediate with 11 of 32 samples from infected trees. This study documents the occurrence of three species of Armillaria in the Arkansas Ozarks and their association with oak mortality resulting from an oak decline event coupled with a red oak borer, Enaphalodes rufulus, outbreak. Further, it documents some potential variation in host/pathogen combinations and forest site conditions.     

Armillaria species in Japan identified by isozyme patterns with special reference to the biological species of the northern hemisphere

Isozyme patterns were examined to characterize biological species ofArmillaria in Japan. Of 25 different enzymes tested, glutamate dehydrogenase (GDH), lactate dehydrogenase (LDH), and aspartate aminotransferase (AAT) showed the most stable and reliable activities. These isozyme patterns showed little variability between haploid and diploid hyphae even under different cultural conditions among isolates tested. From the results, six Japanese biological species were clearly characterized through combination of GDH and LDH isozymes. There were no differences on the whole in isozyme patterns among European biological species ofArmillaria.     

Incidence and phylogenetic analyses of Armillaria spp. associated with root disease in peach orchards in the State of Mexico,Mexico

Incidence of peach [Prunus persica (L.) Batsch] tree mortality attributed to Armillaria root disease was assessed from 2009 to 2011 in 15 orchards in the State of Mexico, Mexico. Incidence increased gradually every year of assessment, reaching average values of 9.7, 15.3 and 20.3% tree mortality and 23.2, 24.7 and 28.3% disease‐impacted area of the orchards during 2009, 2010 and 2011, respectively. The cultivars ‘Nemaguard’ and ‘Criollo of La Goleta’, a local rootstock used in the region, were both susceptible to the disease. To identify species of Armillaria isolated from infected peach trees, two nuclear rDNA regions (partial 5.8S‐ITS2‐LSU D‐domains and partial 3′ LSU‐IGS1) and the translation elongation factor‐1α (tef‐1α) gene were sequenced and compared with sequences of known Armillaria species. DNA sequence analysis from 49 Armillaria isolates revealed that five isolates (10.2%) were Armillaria mellea and eight isolates (16.3%) were Armillaria gallica. DNA sequences from the remaining 36 isolates (73.5%) showed no close similarity to Armillaria sequences in GenBank, and apparently represent an undescribed Armillaria species. This undescribed species was the most widely distributed in the region of study. Separate phylogenetic analyses of the LSU region (D1–D3 domains concatenated with the partial 3′ end) and the tef‐1α region show that the undescribed species is quite distinct from other Armillaria spp. reported in North America.     

Identification of Armillaria species in Japan using PCR-RFLP analysis of rDNA intergenic spacer region and comparisons of Armillaria species in the world

Armillaria species from Japan were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the intergenic spacer region-1 (IGS-1) of ribosomal DNA (rDNA). Eleven different digestion patterns by restriction endonuclease Alu I were found among 70 isolates of seven Armillaria species in Japan. Isolates within Armillaria nabsnona, A. ostoyae, A. cepistipes, and Japanese biological species E showed the same Alu I digestion patterns. Five Alu I patterns were detected for A. gallica, three patterns for A. mellea, and two patterns for A. tabescens. Seven Armillaria species in Japan were clearly distinguished by using the profiles obtained when PCR products were digested with Alu I, Msp I, and Hae III restriction enzymes. There was considerable variability of Alu I restriction sites within the IGS-1 between the isolates of five Armillaria species, A. gallica, A. nabsnona, A. cepistipes, A. mellea, and A. tabescens, in Japan and those of their European and North American counterparts.     

Phylogeography and host range of Armillaria gallica in riparian forests of the northern Great Plains,USA

Root disease pathogens, including Armillaria, are a leading cause of growth loss and tree mortality in forest ecosystems of North America. Armillaria spp. have a wide host range and can cause significant reductions in tree growth that may lead to mortality. DNA sequence comparisons and phylogenetic studies have allowed a better understanding of Armillaria spp. taxonomic diversity. Genetic sequencing has facilitated the mapping of species distributions and host associations, providing insights into Armillaria ecology. These studies can help to inform forest management and are essential in the development of disease risk maps, leading to more effective management strategies for Armillaria root disease. Armillaria surveys were conducted on publicly owned lands in North Dakota, South Dakota, and Nebraska, U.S.A. Surveyed stands consisted of riparian forests ≥0.4 hectares in area. Armillaria was found at 78 of 101 sites. A total of 57 Armillaria isolates—associated with 12 host tree species—were used for DNA sequencing of the translation elongation factor‐1 alpha (tef1) gene. Armillaria gallica was the only species identified within the study sites. Results suggest that A. gallica is a common root pathogen of hardwood trees in riparian forests of the northern Great Plains with a wider host range and geographic distribution than previously recognized.     

Mycelial fan formation of three sympatric Armillaria species on excised stem segments of Picea abies

Mycelial fan formation was studied in five Armillaria cepistipes, ten A. borealis and ten diploid and six haploid A. ostoyae strains on excised stem segments of Picea abies. Stem segments were either non‐autoclaved or autoclaved, representing dying and dead wood, respectively. To confirm the identity of mycelial fans on non‐autoclaved stem segments, re‐isolations were made and isolates characterized with microsatellite markers. Mycelial fan formation on autoclaved stem segments was fast and reliable for most of the tested Armillaria strains. On non‐autoclaved stem segments, mycelial fan formation was slower, more erratic and less predictable. Mycelial fan formation was fastest in A. cepistipes closely followed by A. borealis and was slowest in A. ostoyae. For two A. cepistipes and four A. ostoyae strains (all diploid), growth rates of mycelial fans were estimated in a time course experiment. They ranged between 5.1 and 8.7 mm/day for autoclaved and between 1.4 and 4.7 mm/day for non‐autoclaved stem segments. The haploid A. ostoyae strains also formed mycelial fans on autoclaved stem segments, but typically slower and less reliably than the diploid strains. Whether haploid strains are able to produce mycelial fans on non‐autoclaved stem segments remains unknown because of accidental diploidization of the original haploid strains which was likely caused by basidiospores introduced into the study system on the non‐autoclaved stems. Overall, the method developed in this study may be useful for further investigations into the genetic, physiological and biochemical nature of mycelial fan formation in the genus Armillaria.     

Phenotypic characterization of cryptic Diplodia species by MALDI‐TOF MS and the bias of mycelium age

Detection and identification of fungal cryptic species has been facilitated by DNA sequencing. However, the examination of some phenotypic traits is fundamental for the confirmation of genetic results. MALDI‐TOF mass spectrometry has shown remarkable reliability in the recognition of species‐specific phenotypic markers and has already been tested in several species of fungi. However, even though there is no direct evidence, the age of the mycelium appears to influence the composition of the metabolites detected. In this study, we demonstrate that MALDI‐TOF is a reliable technique to identify suitable metabolites to distinguish three monophyletic species of Diplodia (D. pinea, D. seriata and D. scrobiculata), thus supporting recent DNA results. Nevertheless, different collections of samples over a period of 3 months after inoculation also revealed that the MALDI‐TOF spectra are highly dependent on mycelial age. More importantly, the species‐specific markers emerge only after 1 month of mycelial growth. The methodological and biological implications of these findings are discussed.     

Single sequence repeat markers reflect diversity and geographic barriers in Eurasian populations of the conifer pathogen Ceratocystis polonica

The blue‐stain fungus and vascular stain pathogen Ceratocystis polonica and its associated bark beetle vectors, particularly Ips typographus and I. typographus japonicus, cause significant losses to several spruce species in Eurasia. Nothing is, however, known about the population genetics of this conifer pathogen. In this study, a set of single sequence repeat (SSR) markers were developed to determine the population structure and genetic diversity of C. polonica in Europe and Japan. ISSR‐PCR primers were used to target SSR‐rich regions and specific primers were designed flanking the SSR regions found in these amplicons. The SSR primers developed for C. polonica were found to be transferable to six other Ceratocystis species from conifers, residing in the Ceratocystis coerulescens complex. Ninety‐eight isolates representing four populations of C. polonica (Austria, Norway, Poland and Japan) were tested using 10 selected polymorphic SSR markers. A high level of gene diversity was found in C. polonica as a whole (H = 0.53). Analysis of G statistics showed a low degree of population structure in Europe and a high level of gene flow between populations (Gst = 0.05, Nm = 8.5). In contrast, the Japanese and the European populations of C. polonica displayed strong genetic separation, which is likely caused by geographic isolation. The low level of population structure of C. polonica in Europe and the differentiation between the European and the Japanese fungal populations mirror previous findings for I. typographus and I. typographus japonicus, the main insect vectors of this fungus. These results support the view that the fungus and the insect have closely co‐evolved together. This study also suggests that movement of C. polonica and its vectors between Europe and Asia pose a threat to forestry on both continents and this should clearly be avoided.     

Direct genotyping of the poplar leaf rust fungus,Melampsora medusae f. sp. deltoidae,using codominant PCR‐SSCP markers

Two anonymous DNA markers that are revealed by single‐strand conformational polymorphism (SSCP) analysis were developed for detection of polymorphisms in Melampsora medusae f. sp. deltoidae (Mmd). Mono‐uredinial isolates of Mmd were first obtained, DNA was extracted from urediniospores and random amplified polymorphic DNA (RAPD) products of eight mono‐uredinial isolates were separated on a SSCP gel to identify differences among them. Bands representing putative polymorphic loci among the eight isolates tested were excised from the SSCP gel and re‐amplified by polymerase chain reaction (PCR), and then cloned and sequenced. A primer pair was designed to amplify a DNA fragment of a size suitable for SSCP analysis (<600 bp) for two out of three DNA fragments sequenced. Each set of primers amplified a PCR product for all eight isolates that were initially used to generate them and the resulting PCR products were analysed by SSCP. Polymorphisms among isolates were identified for both putative loci. The two primer pairs amplified a PCR product of the expected size on an additional 32 mono‐uredinial isolates of Mmd tested. From the overall 40 mono‐uredinial isolates tested, 5 and 11 alleles were detected, and 12 and 34 isolates showed to be heterozygous, as indicated by the presence of more than two bands on the SSCP gel, at loci A and B, respectively. The primer pairs were tested for specificity against 106 fungal isolates belonging to various taxa, including other rusts, and against DNA extracted from greenhouse‐grown healthy poplar leaves. DNA amplification products of the expected size were obtained only when Mmd DNA was present. Optimization of PCR conditions with these two primer pairs allowed genotyping directly from single uredinia extracted from infected leaves, thus alleviating the need to culture the fungus to characterize individuals, hence making it possible to process large numbers of samples for population studies.     

Ecology and distribution of Armillaria species in Norway

The occurence of Armillaria species was assessed in Norway, enabling the northern‐most distribution of this genus to be determined in Europe. Four Armillaria species were found in Norway. Armillaria borealis was the most common species occurring on woody vegetation to the permafrost zone (ca. 69°N). Armillaria cepistipes was present in southern and central Norway, but was not found further than 66°N. Armillaria solidipes and Armillaria gallica were rare, found at only one locality each; 59°40′ and 59°32′, respectively. Armillaria species were found on 14 hosts, but there was no significant difference between occurrence of A. borealis and A. cepistipes on declining and dead trees. Phylogenetic analyses separated each species into separate clades. All isolates of A. borealis, except one, and most isolates of A. solidipes were in separate clades. However, a subclade within the A. borealis clade was formed of two A. ostoyae and one A. borealis isolates. Two small A. cepistipes genets were found in a declining oak stand.     

Development of microsatellite markers for Thekopsora areolata,the causal agent of cherry spruce rust

Cherry spruce rust is a fungal disease of Norway spruce cones caused by Thekopsora areolata and responsible for significant losses in seed production in Sweden and Finland. Here, we report the first set of nine microsatellites, which will allow an effective genetic fingerprinting of T. areolata. The markers were isolated using the FIASCO method and were characterized using DNA from 49 single aecia sampled from spruce cones in three different seed orchards in Sweden. Eight of the nine markers were shown to be polymorphic among the aecia. The markers were unlinked and are therefore suitable for future population genetic studies.     

Ecology of Armillaria species on conifers in Japan

Distribution, host preference and pathogenicity of Japanese Armillaria species on conifers were investigated on the basis of field collections of 65 isolates. We identified seven Armillaria species from 19 conifer species including six major Japanese plantation conifers using mating tests and sequences of the translation elongation‐1 α gene. Armillaria mellea, Armillaria ostoyae, Armillaria cepistipes and Armillaria sinapina were frequently collected, whereas Armillaria nabsnona, Armillaria tabescens and a biological species Nagasawa’s E were rare. On the basis of host condition when the isolates were collected, A. mellea, A. ostoyae, A. cepistipes and A. tabescens are considered as moderate to aggressive pathogens of conifers in Japan.     

Ecology of Armillaria species in managed forests and plantations in Serbia

The distribution of Armillaria species was investigated in Serbian forest ecosystems, in relation to the main host species attacked, forest‐types, geography and altitude. In total, 388 isolates were identified from 36 host species in 47 sites. Armillaria gallica was the most commonly observed species with the widest distribution and with an altitudinal range of 70–1450 m, it was the dominating Armillaria species in lowland alluvial forests and in Quercus and Fagus forests at higher elevations. Armillaria mellea occurred in Quercus spp. – dominated forests in the north and central regions at 70–1050 m. Sixty‐eight per cent of the A. mellea isolates were collected from living hosts, most commonly in declining conifer plantations. Armillaria ostoyae was distributed in the cooler coniferous forest types and plantations in the Dinaric Alps in the south of Serbia, at 850–1820 m. Armillaria cepistipes was found in the eastern and southern hilly and mountainous regions of the country, at 600–1900 m. Most isolates were obtained from conifers and rhizomorphs in the soil around decaying stumps. Armillaria tabescens was found only on dead oak material in the northern and eastern regions of the country at altitudes lower than 600 m.     

Molecular‐based identification and phylogeny of Armillaria species from Serbia and Montenegro

Armillaria causes problems of root rot, kill trees and decay wood in the forests of Serbia and Montenegro, but the species involved have not hitherto been identified. The aim of this study was to identify field isolates collected on 25 localities. Identification was based on restriction fragment length polymorphism (RFLP) analysis of intergenic spacer 1 (IGS1) region and comparisons of IGS1 sequence with those available on NCBI database. Phylogenetic analysis was performed on sequence information from selected isolates to determine possible interrelationships between isolates with different banding patterns and previously identified tester isolates of five European Armillaria species. Five Armillaria species were identified in 90 isolates obtained from forests in Serbia and Montenegro. Armillaria gallica was most frequently isolated, followed by A. cepistipes, A. mellea, A. ostoyae and A. tabescens; two isolates remained unidentified. Restriction digestion of IGS1 amplification products with AluI produced 10 RFLP patterns. Patterns G4 (400, 250, 180) for A. gallica and pattern X (400, 180, 140) for isolates 74 and 79 are reported for the first time in European isolates. Eight RFLP patterns were observed after restriction with TaqI. Two patterns each were observed for A. ostoyae and A. gallica, and one each for A. cepistipes, A. mellea, A. tabescens and isolates 74 and 79. Parsimony analyses based on the IGS1 region placed the isolates into four clades: one including A. mellea, the second containing A. gallica–A. cepistipes isolates, while isolates of A. ostoyae and A. borealis were in the third clade. Armillaria tabescens differed from all annulate species. Phylogenetic analysis supported the conclusion that European Armillaria species are closely related and separated from a common ancestor in the near past. According to this survey five European Armillaria species are present in the forests of Serbia and Montenegro, while A. borealis is not present in the studied ecosystems.